1A Standard Parts List for Biological Circuitry
Background and Overview
One of the hallmarks of biochemical circuits found in nature is analog, asymmetric, asynchronous design. That
is, there is little standardization of parts, e.g. all the promoters have different strengths and kinetics,
transcription factors are designed to have different effects at different loci, and each enzymatic reaction has its own
idiosyncratic mechanism and rates. In addition, all of the heterogeneous circuit elements are executing their
functions concurrently and asynchronously. Biological circuits are seemingly designed to deal with the
fluctuating delays, different time-scales and energy requirements associated with each component process of the
overall network. These factors also make design of novel biochemical circuitry from existent parts difficult to
achieve. Without standardization, the qualitative design methods used in other engineering fields are simply
inapplicable. The de facto design methodology for biological circuitry is natural selection. Rational design of
biological systems by humans has remained restricted to rather small or hit-or-miss efforts and has often relied
on the ability to "select" for biochemical parts that fulfill some criteria. In practice however biological-designers
are rare, and solutions are usually realized through an expensive stepwise trial and error approach or through
mutation and selection. Furthermore, these otherwise practical approaches are limited in terms of the problems
they can solve. We believe that implementation of designed biological circuitry is limited by issues of practice.
We believe that implementation of designed biological circuitry is, in large part, limited by issues of practice.
To address this deficiency, we propose herein a program to produce a set of well-characterized and systematized
biological components that can be generically assembled to create custom biological circuitry. Our goal is to
lead to a toolkit of biological reagents that can be assembled into biological circuitry in the same way resistors,
capacitors, and transistors can be assembled to make electronic circuitry.
This proposal has four specific aims:
1. The selection, refinement, and standardization of existing biological components such that they
become useful for implementing biological circuitry,
2. The need-driven creation of novel components,
3. The extensive characterization of each component's "device physics" sufficient to allow the reduction to
component input/output in design simulations of complex circuitry.
4. The construction of a small set of test circuits to demonstrate the efficacy of the part design.
The material deliverables here are a set of "pluggable" reagents that may be assembled into circuits. By
"pluggable" we mean that are designed in such a way that they may be particularized by the clipping in of
specific DNA sequence (either genes or regulatory regions). A good example of a "pluggable" system already in
general use is the two-hybrid system that allows the "plugging-in" of two test proteins into specially designed
vectors that, in the properly engineered strain of yeast, can test for protein-protein interaction. We envision
using constructs of similar spirit to attach well-known degradation or localization signals to proteins, or which
reverse the two hybrid experiment to allow fusion of two proteins (say a kinase and response regulator that do
not naturally interact) to well-known interaction domains. Similarly, we envision constructs that allow the
specific engineering of the translational efficiency and degradation rate of RNA molecules by the "pluggable"
swapping of 3' and 5' untranslated regions (UTRs). We describe in a little more detail a variety of such
constructions below. The characterization of the reagents and the input/output simulation models of these
reagents are also deliverables of this proposal.
It is imperative, that we reach a level of engineering facility with these systems comparable to that we have for
mechanical and electronic systems if we are to achieve a number of central industrial and medical goals. There
are major industrial efforts in pathway engineering of plants, animals and bacteria for production of biochemicals
for pharmaceutical and agricultural needs, for bioremediation and for improvement of the organism itself as an
agricultural product. These efforts currently suffer from the lack of well characterized
We will confine ourselves initially to Escherichia coli (E. coli) and Saccharomyces cerevisiae (yeast), as our test
beds for our constructs. These two organisms are central model organisms as well as being of industrial interest.
In addition, organisms have unique properties that make them more or less amenable to cellular engineering.

2Refinement of Existing Components and Benchmarking
As the fields of biochemical and genetic engineering are already well established, a major part of our proposal
involves the collection, characterization, and systematization of existing biological components. What makes
this useful is the level of component standardization and experimental characterization we will apply throughout
the process. Below are described some of the components we will initially pursue. We will focus in the course
of this proposal only on those components that aid in building our test circuits. As time and need progresses
we will collect and engineer more the components we discuss below. The basic idea in all cases will be to
produce set of vectors that allow engineering at three levels of biochemical circuitry: transcriptional control,
translational control and post-translational reactions. Diagrammatically, there are a number of regions in a vector
that we define. We show the eukaryotic case because the prokaryotic case is a defined subset.
Transcriptional Design Components
Components of this type allow for the regulation of transcription. Engineering of these component parts
concerns boxes, 3,4, and 9 on the diagram above. We have identified a number of existent components that may
be useful in creating standardized reagents with specific transcriptional activities:
Promoter Control Engineering
The section concerns engineering of box 3 on the above diagram and the collection of proteins that can interact
with elements within box 3. The elements within box 3 include RNA polymerase binding sites and operator
regions (or upstream activating, repressing sequences, U(A,R)Ss). The goal here is to create a set of well-
characterized transcription initiation control "gates" with different molecular specificities and activities such that
complex circuits can be built from them.
1. We propose to collect a set of fixed orthogonal RNA polymerase-promoter series. Orthogonal in the
sense that the polymerase from one series does not recognize promoters from another. Series in the
sense that each polymerase-promoter pairing is available over a range of activation levels created by
mutation in the promoter and/or polymerase. Fixed in the sense that variation in activity requires
genetic modification of either the polymerase or the promoter,[1, 2]
2. These will be complemented by a set of variable orthogonal RNA polymerase-promoters. As above
except that the activity of each RNA polymerase-promoter pairing is subject to the level of an
activating or repressing species. [2-5]
3. For yeast constructions, we will collect a set of endogenous and exogenous transcription factor binding
sequence for direction of protein to the promoter (e.g. synthetic gal4 UAS, lexA binding motif,
repressor binding motif [6]) and a set of promoters that are active under different conditions, such as
3. Promoter Control
4. 5’ UTR
5. N’-end fusion
6. Gene
7. C-end fusion
8. 3’ UTR
9. Term./IRES
5’ UTR
2. Proteotrophy
1. Resistance Cartridge
Not all vectors will have all components. Different vectors will have different purposes (fusion
construction, expression, etc.). These vectors will be engineered with compatible restriction sites
between regions of interest. We break engineering vectors into regions as follows:
For passage between, E. coli and yeast, for maintainance of multiple plasmids in E. coli
To allow selection in yeast, different proteotrophies allow maintenance of multiple plasmids in yeast
To allow selection of polymerase and transcription factor specificities
For engineering of RNA translational efficiency and stability (e.g. stem-loops, antitermination sites)
For engineering the N-end of particular proteins (e.g. to allow degradation or localization signals)
Insertion point for protein of interest.
C-end fusion protein (e.g. for fusion of domains with specific activity)
To allow engineering of RNA stability
To allow engineering of elongation control elements. (IRES= internal ribosome entry site)
Next RNA or protein gene...